Assay multiplexing is of great interest and utility in the biomedical field. In particular, there is a need to measure multiple analytes in a given sample. This leads to analysis of more information for a particular sample, allowing greater utility in the diagnosis and identification of important biological markers in a given sample.
Bead-based multiplexing of bioassays is a common approach to sample multiplexing. For instance, it is known to use fluorescent beads that are dyed for multiplexing based on the amount of dye in the bead for identifying the sample. A bead that has low fluorescence in one color and high fluorescence in a second color can be readily distinguished in a multiplexed manner. The second color can then be utilized for measurement of the desired analyte.
Other permutations of bead-based multiplexing include the use of quantum dots for coding multiple colors in a microparticle. In this approach, quantum dots, which have sharp spectral emission profiles, can be mixed together in a microsphere. Ratios of the spectral emissions of the various colors allow for identification of the particle of interest. Since quantum dots have narrow emission profiles, several colors can be mixed together to allow for a significant level of multiplexing.
Hydrogel microparticles have been multiplexed by the addition of a simplified barcode to the header of the microparticle. Hydrogel microparticles can also have multiple elements, by where intensity coding of the uniform elements allows for multifunctional coding. Both barcodes on the header and within the microparticles lead to a unique intensity versus time profile signature on every microparticle.
Other types of coded microparticles include sub-micrometer metallic barcodes, 2D hydrogel barcodes, and one-dimensional barcodes. Glass microparticles can be fabricated to generate a 1D diffraction pattern that uniquely identifies the particles. Most of these methods require sophisticated imaging and decoding algorithms.
Hydrogel microparticles are well suited for biological assays and multiplexing. For instance, PEG microparticles have low autofluorescence, are porous, and can have different functionalities. In particular, PEG with reactive acrylate groups can be utilized to form hydrogel particles using ultraviolet (UV) exposure. PEG microparticles thus have desirable attributes for biological applications. PEG microparticles are advantaged over conventional polymeric (polystyrene, latex, etc.) particles in that they are porous, have low autofluorescence, and low non-specific binding.
PEG particles are readily formed by UV curing of acrylate functionalized PEG. PEG-monoacrylate (PEGMA) and PEG-diacrylate (PEGDA) are common forms of UV curable PEG. This involves the use of a photoinitiator with an acrylate functionalized PEG mixture. Exposure to UV leads to the initiation of the reaction and thus the formation of the particles.
Monodisperse PEG microspheres can be formed by droplet generation inside a microfluidic device. Hexadecane, which is immiscible with the PEG solution, is utilized as the sheath and a mixture of PEGDA and photoinitiator is the aqueous phase. The surface tension of the PEGDA mixture leads to formation of microspheres as the PEGDA stream is focused by the hexadecane phase. A surfactant in the hexadecane helps control the size of the droplets being formed. The hexadecane and PEGDA solutions are in a polydimethylsiloxane (PDMS) microfluidic device. The droplets are photocured by UV light to form stable particles that can be collected and utilized for downstream applications.
Diverse planar PEG microparticles can be synthesized by stop-flow or continuous flow lithography. In this approach, the shape of the PEG particles is controlled by a photomask placed at the field stop position of the microscope. UV light passing through the photomask is projected onto a flowing stream or stopped stream of PEGDA and photoinitiator mixture. The solution is cured by the UV light in the shape of the photomask. Different shapes have been fabricated: barcodes, circles, triangles, and rectangles. One of the advantages of this approach is that the shape is directly controlled by the photomask. Furthermore, the particle size and shape will be uniform. The throughput of particle production, however, will be lower than that of the droplet formation method. An additional advantage of PEG microparticles synthesized by either stop-flow or continuous flow lithography is that multiple laminar flow streams can be co-polymerized together to form multiple elements.
PEG microparticles on glass slides have also been synthesized. These PEG microparticles are formed by placing a droplet of the PEGDA and photoinitiator solution on a PDMS-coated glass slide and then exposing to UV light through a photomask. The shape of the photomask is projected onto the solution on the PDMS slide. The polymerized particles are flushed from the slide. This approach avoids the need for microfluidic channels, pumps, and fluid control. It represents a very simple method of producing uniform microparticles.
The flexibility of hydrogel particle fabrication lends itself readily to multiplexing. Because of this, numerous complex methods employing 1D and 2D can be readily implemented, but these may not be the best or most simple solutions. While 1D and 2D barcodes can lead to high levels of multiplexing, in practice, this is not necessarily required. Based on the complexity of these approaches, there is a need for increased simplicity in multiplexing.